ABSTRACT
Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different concentrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated markers CD90 and CD105. When the concentration of nicotine was 10⁻⁴ mol·L⁻¹, the PDLSC proliferation could be suppressed after 3 d compared with the control group (P<0.05). The amount of mineralized nodules reduced after osteogenic differentiation at 21 d by alizarin red staining. RT-PCR and Western blot showed the expression levels of alkaline phosphatase (ALP), and osteocalcin (OCN), and the Runt-related transcription factor-2 (Runx-2) were lower than in the control group when nicotine suppressed the PDLSCs (P<0.05). This effect was attenuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression patterns of osterix in the early stage of cranio-maxillofacial developmental in zebrafish and to prepare for a further research of osterix gene in bone and tooth development.</p><p><b>METHODS</b>The osterix templates were amplified by PCR to generate DIG labeled antisense and sense probes. Whole mount in situ hybridization was used to analyze the expression patterns of osterix in the early stage cranio-maxillofacial development of zebrafish. The expression patterns of osterix gene in mineralization progresses of cranial and maxillofacial bones were compared. The osterix gene expression in tooth development and mineralization was highlighted by alizarin red staining.</p><p><b>RESULTS</b>Specific DIG labeled probes of osterixwere synthesized successfully. The whole mount in situ hybridization showed that the osterix expression was in the intramembranous ossification at 3 days post fertilization(dpf) and 4 dpf. The specific osterix expression in tooth at 5 dpf and 6 dpf were also observed. The sense probe served as a negative control. Osterix expressed in the unmineralized early bone matrix, the tooth matrix of the primary tooth(3V(1), 5V(1)) and the first replacement tooth(4V(2)).</p><p><b>CONCLUSIONS</b>Our findings showed that osterix might play roles in the process of the early mineralized bone matrix changing into the late mature mineralized bone matrix and the process of development and mineralization of tooth crown matrix.</p>
Subject(s)
Animals , Calcification, Physiologic , Genetics , Gene Expression , Gene Expression Regulation, Developmental , In Situ Hybridization , Maxillofacial Development , Genetics , Osteogenesis , Genetics , Sp7 Transcription Factor , Tooth , Metabolism , Transcription Factors , Genetics , Metabolism , Zebrafish , Zebrafish Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the alteration of interleukin-17 and interferon-γ double positive cells (IL-17(+)/IFN-γ(+) cells) in mice with coxsackie virus B3 (CVB3) induced acute viral myocarditis (VMC).</p><p><b>METHODS</b>VMC was induced in male Balb/c mice by peritoneal injection of CVB3. Control mice received PBS injection. At 0, 1, 2, 3, 4 and 6 weeks after injection, pathological scores were determined on hematoxylin-eosin stained heart sections and flow cytometric analysis was performed to evaluate the percent of IL-17(+)/IFN-γ(+) cells among CD4(+) T cells.</p><p><b>RESULTS</b>Compared to control mice, the pathological scores of VMC mice were higher on CVB3 infection week 1 (1.8 ± 0.5), peaked on week 2 (2.8 ± 0.5) and declined thereafter. However, the proportion of IL-17(+)/IFN-γ(+) cells remained steadily at a low level throughout the observation period and was similar between VMC and control mice.</p><p><b>CONCLUSIONS</b>Our data shows that IL-17(+)/IFN-γ(+) cells might not be involved in the inflammation process of CVB3 induced VMC mice model.</p>